1. Field of the Invention
The present invention relates generally to analysis of extracellular fluids that contain carrier proteins (e.g., serum albumin), and, more particularly, but not by way of limitation, to methods for prognosis and detection of active (e.g., aggressive) malignancy in a patient by analyzing serum or plasma samples from the patient.
2. Description of Related Art
For illustration, but without limiting the scope of the invention, the background is described with respect to analyzing the blood of a human patient.
In the patient with active cancer, malignant cells may grow aggressively, forming and growing malignant cells and invading parts of the body near an initial tumor during early stages, and spreading to more distant parts of the body through the blood stream and/or lymphatic system during later stages. These processes are often accompanied by secretion of multiple metabolites to the circulatory system of the patient.
Analyzing hematologic parameters and/or measuring the concentration of various metabolites in blood samples from a patient are known in the art and may be widely-used methods of diagnosing cancer in a patient, such as, for example, by identification of antigens, hormones, enzymes and other biologically active substances (tumor markers).
Currently known tumor markers are generally each useful for diagnosing a certain type of cancer, such as, for example, alpha-fetoprotein for liver cancer, human chorionic gonadotropin (HCG) for some types of testicular and ovarian cancer, prostate-specific antigen for prostate cancer, CA 15-3 for breast cancer, and several others. These tumor markers may not permit recognition or diagnosis of active cancer until later stages when other symptoms are already noticeable and the patient is investigated with methods such as X-rays, CT scans, endoscopy and biopsy. In these later stages, treatment may be less effective and may not be effective enough to prevent the death of the patient.
One example of a known method may be referred to in the art as “mass spectrometry-based profiling” of metabolites residing in extracellular fluids (also known as Metabonomics), such as, for example, profiling of low molecular mass serum proteins (Proteomics) and peptides (Peptidome). Certain studies have indicated that, in a patient with any of several types of types of active malignancy, mass-spectrometry based profiling may detect significant changes in profiles of low-molecular mass metabolites in serum and other extracellular fluids. These changes appear to be caused by (i) infusion of specific metabolites produced by cancer cells, (ii) disturbance of normal metabolic processes resulting from mediators, intermediates and other bio-active molecules secreted by tumor cells, and/or (iii) acute-phase response metabolites produced by tissue invaded by malignant cells. Certain of these studies also discovered that low-molecular weight metabolites (e.g., peptide fragments) that may be generated as a consequence of the disease process, are often immediately bound to high-abundance carrier proteins (e.g., albumin), and thereby prevented from being cleared from the patient's blood by the liver.
Serum albumin is generally the most abundant serum protein, and provides a primary transport of hydrophobic metabolites in the circulatory system of the organism. The structure of the albumin molecule includes three domains that, in the hydrophilic medium of blood, are generally folded in an albumin globule of a heart-like and/or ellipsoidal shape in which hydrophilic binding sites generally face outward, and the hydrophobic binding sites generally face inward to form what may be referred to as one or more “hydrophobic containers” or “hydrophobic cavities” in the interior of the globule. The albumin molecule has multiple specific and non-specific hydrophobic sites which bind various long-chain fatty acids and other hydrophobic metabolites. In the albumin-bound state, hydrophobic metabolites are transferred and/or accumulated in hydrophilic extracellular fluids in the body.
Known techniques of profiling of serum metabolites generally require dissociation of all albumin-bound metabolites, which may cause a number of shortcomings and/or drawbacks. Additionally, known techniques for profiling of serum metabolites generally are as a research tool for discovery of new tumor biomarkers, rather than as a diagnostic tool for diagnosing cancer.
European Patent Application, EP 0 973 043 A1, published Jan. 19, 2000, describes a method of diagnosing malignant neoplasms by using electron paramagnetic resonance (EPR) spectroscopy (which may also be known in the art as electron spin resonance “ESR” spectroscopy), wherein an aliquot of a subject patient's serum is mixed with the spin probe represented by spin labeled fatty acid. In this method, a malignant disease is detected based upon the deviation of physicochemical parameters of the mobility of the spin probe in three binding sites of serum albumin, relative to values exhibited in healthy subjects. This method may produce false-positive and false-negative results in the presence of additional factors such us evaluated blood lipid values, injection of medicaments, elevated blood alcohol level, residing infection diseases of the subject patient, as well effects caused by a solvent of the spin probe.
U.S. Pat. No. 7,166,474 describes a method of detecting changes in transport properties of albumin by using electron paramagnetic resonance (EPR) spectroscopy (which may also be known in the art as electron spin resonance “ESR” spectroscopy) to evaluate a sample that contains albumin (an albumin-containing sample). This method can include evaluating the albumin transport function with respect to long chain fatty acids by using a spin probe represented by a spin-labeled fatty acid. Specifically, according to this method, the EPR-spectra of the spin probe can be measured in at least three aliquots of the sample, where each aliquot is mixed with significantly different concentrations of the spin probe and a high concentration of ethanol. The concentration of ethanol is high enough to induce significant conformational changes of the albumin to enable evaluation of the conformational flexibility of the albumin. The conformational state and binding parameters of the albumin are determined from the parameters of the spin probe mobility in two binding sites of serum albumin, and the concentration of unbound spin probe. The parameters of albumin transport function are derived from measurements of the conformational changes induced in the albumin artificially by the high ethanol concentration. This method assumes that albumin molecules efficiently release bound substances to target objects at conditions that occur in a healthy patient, and therefore induces a conformational change facilitating dissociation of albumin-bound metabolites.
This method can include detection of cancer via the evaluation of the conformational changes of albumin in respect of modification caused by metabolites associated with a tumor growth. While this may be useful in the method taught by this patent, the conformational changes of albumin molecules induced by the high ethanol concentration cause dissociation of a significant portion of albumin-bound spin probe in the investigated sample, which largely prevents correct evaluation of the albumin conformation. For example, due to their hydrophobic origin, spin probe molecules dissociated from albumin will aggregate into micelles and associate with or bind to serum lipoproteins. During the measurements of EPR spectrum, these dissociated portions of spin probe (aggregated into micelles and associated with or bound to serum lipoproteins) can induce EPR signals that interfere with those from the spin probe bound on albumin sites, and thereby prevent precise evaluation of the albumin conformation.
This method suffers from possible shortcomings that may include, for example, the use of at least three aliquots of each sample, binding on albumin of acute-phase response metabolites in early (e.g., asymptomatic) stages of infection or disease, and excessive dissociation of spin probe from the albumin (and resulting spin probe aggregation into micelles and binding to serum lipoproteins) resulting from the addition of relatively high-concentrations of ethanol. These possible shortcomings may, in some instances, contribute to variations in results, including false-positives and false-negatives in detecting the presence of metabolites associated with an active malignant growth in a patient.